E-selection binding soluble lamp-1 polypeptide

ABSTRACT

The present invention provides novel purified human lysosomal membrane sialoglycoproteins. These novel human proteins, lamp-1 and lamp-2, are highly glycosylated and are the major carriers of polylactosaminoglycan, when expressed on the cell surface participate in various cellular adhesion interactions.

This invention was made in part with Government support under Grant Nos.R01 CA48737 and P01 AI33189 from the National Institutes of Health. TheGovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Adhesive interactions of cells with other cells and with theextracellular matrix are crucial to all developmental processes, buthave a central role in the functions of the immune system throughoutlife. Leukocyte trafficking, recruitment to sites of inflammation, tumorcell adhesion to endothelial cells and metastasis are mediated by threeadhesion receptor families, the integrin and immunoglobulinsuperfamilies and the recently described selectin family. The knownselectins contain an N-terminal lectin domain that mediates adhesion bybinding carbohydrate ligands on opposing cells. The lectin domain isfollowed by an epidermal growth factor-like domain and a series ofconsensus repeats similar to those found in complement regulatoryproteins. The selectins are expressed on activated endothelial cells andplatelets and are implicated in the recruitment of neutrophils andmonocytes to sites of tissue injury.

E-selectin is a selectin that is transiently expressed on endothelialcells 2-8 hr after stimulation of IL-1 and other inflammatory agents,and mediates a neutrophil adhesion pathway distinct from that mediatedby ICAMS and leukocyte integrins. The neutrophil chemoattractant IL-8,which is secreted by activated endothelial cells, acts on neutrophils asa feedback inhibitor to attenuate the hyperadhesive interaction betweenneutrophils and E-selectin receptors. P-selectin is located inα-granules of platelets and Weibel-Palade bodies of endothelial cells,and is rapidly mobilized to the surface of these cells after stimulationby products of the clotting cascade such as thrombin, where it mediatesadhesion of neutrophils and monocytes. Selectins function in a widerange of cell interactions in the vasculature and are expressed both onleukocytes and endothelial cells. Selectins mediate adhesion eventswithin the blood vascular compartment through calcium-dependentrecognition of specific carbohydrates.

The acquisition of invasive properties by tumorigenic cells constitutesan essential step in tumor progression. Since most malignant tumors arecarcinomas, the molecular mechanisms underlying the invasion ofepithelial cells are of particular interest.

Over 90% of human tumors are carcinomas; in these, transformedepithelial cells grow in an uncontrolled fashion, break through thebasement membrane, and invade the underlying mesenchyme. Local invasioncan compromise the function of involved tissues. It has been shown thatthe state of differentiation and the concomitant degree of invasivenessof carcinomas can determine cancer progression. However, the mostsignificant turning point in the disease is the establishment ofmetastasis. It is known that the malignant phenotype is the culminationof a series of genetic changes that involves both positive and negativeregulatory elements. Investigation of the activation, regulation,mutation, or somatic deletion of genes that encode these regulatoryelements presents a new frontier for research into the complex cellularinteractions that precede the development of metastasis.

The morphological and functional characteristics of carcinomas wererecognized years ago; the underlying molecular basis, however, is onlypresently accessible to the investigation on a molecular level. Thus,there is a great clinical need to elucidate the underlying molecularbasis of cellular adhesion and its role in inflammatory responses andmetastasis and to develop compounds that can modify these cellularinteractions. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides novel purified human lysosomal membranesialoglycoproteins. These novel human proteins, lamp-1 and lamp-2, arehighly glycosylated and are the major carriers of polylactosaminoglycan.Lamp-1 and lamp-2 proteins are expressed on the cell surface participatein various cellular adhesion interactions.

Further provided by the present invention are methods of modifyingbiological functions mediated by the regulatory activity of selectinreceptors, methods of alleviating pathologic conditions mediated bylamp-derived polypeptide and selectin receptor interactions. Isolatednucleic acids encoding the novel lamp-1 and lamp-2 glycoproteins andsoluble lamp-derived polypeptides are provided, as well as vectorscontaining the nucleic acids and recombinant host cells transformed withsuch vectors. This invention provides antisense oligonucleotides capableof binding specifically to mRNA molecules encoding human lamp-derivedpolypeptides. The present invention provides monoclonal antibodies tothe soluble lamp-derived polypeptides. Methods of detecting the presenceof activated selectin receptors on platelets and endothelial cellsurfaces are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D shows the results of flow cytometry analysis of cellsurface lamp-1 on various SP cell lines. From the left, the solid linesrepresent profiles obtained by staining SP cell transfectants withoutprimary anti-human lamp-1 antibody. Dotted lines represent profilesobtained by staining control, non-transfected SP cells with anti-humanlamp-1. The filled profiles were obtained when SP cell transfectantswere stained with anti-human lamp-1 antibody. FIG. 1A, SP celltransfectant obtained with pSVlamp-1. FIG. 1B, SP cell transfectantobtained with pSRαlamp-1. FIG. 1C, SP cell transfectant obtained withpSVlamp-1^(H). FIG. 1D, SP cell transfectant obtained withpSRαlamp-1^(H).

FIG. 2A shows adhesion of various SP colonic carcinoma cells toendothelial cells or E-selectin expressing CHO cells. SP cellstransfected with lamp-1 expression vectors (pSVlamp-1, pSRαlamp-1,pSVlamp-1^(H), or pSRαlamp-1^(H)), or the parental SP cells were used inadhesion assays as described in the Examples. Adhesion toIL-1β-activated HUVEC monolayers is indicated by the open bars, whereasadhesion to non-activated HUVECS is represented by the solid bars.Adhesion to E-selectin-expressing CHO cell monolayers is denoted by thehatched bars, and the cross-hatched bars depict adhesion to control CHOcell monolayers. Data shown correspond to the fraction of applied cellsthat remained after washing, and represent the mean and standarddeviation derived from four replicate assays.

FIG. 2B shows the inhibition of adhesion to endothelial cells by sialylLe^(x) glycolipid. The SP cell line transfected with the pSRαlamp-1^(H)vector was subjected to adhesion to activated HUVEC monolayers exactlyas in panel A (open bar), or as in panel A after pre-treatment of theactivated monolayers with liposomes containing sialyl Lewis^(x)glycolipid (hatched bar), or after pre-treatment with liposomescontaining the control glycolipid paragloboside (cross-hatched bars).Data correspond to the fraction of applied cells still adherent afterwashing, and are the mean and one standard deviation, from fourreplicate assays.

FIG. 3 shows binding of anti-sialyl Le^(x) antibody to various SP-cells.Increasing concentrations of monoclonal antibody specific to sialylLe^(x) were incubated with cells, and binding was determined asdescribed in the "Examples." Cells tested are SP cells transfected withpSVlamp-1 (▴), pSRαlamp-1 (Δ), pSVαlamp-1^(H) (), pSRαlamp-1^(H) (∘),and the parental SP cells (□). All of the transfected cells are the sameas shown in FIG. 1.

FIGS. 4A and 4B show the purification of soluble lamp-1 generated fromcontrol CHO cells and CHO cells expressing sialyl Le^(x) structures. Theculture medium from the CHO cells (lanes 1,4) are successively appliedto wheat germ agglutinin columns (lanes 2,5) and DEAE-Sephadex column(lanes 3,6). Lanes 1-3 are the samples isolated from the control CHOcells while lanes 4-6 are the samples isolated from the CHO cellsexpressing sialyl Le^(x) structures. FIG. 4A is the protein stainingwhile FIG. 4B is the Western blot by anti-lamp-1 antibodies.

FIG. 5 shows the inhibition of cell adhesion to HUVEC by soluble lamp-1.The adhesion of SP cells transfected with pSRαlamp-1^(H), the same asshown in FIG. 1, was tested for inhibition by soluble lamp-1 derivedfrom sialyl Le^(x) positive CHO cells (hatched bars). The controlsoluble lamp-1 was obtained from control CHO cells that do not expresssialyl Le^(x) (cross-hatched bars). The amount of soluble lamp-1 isexpressed as μg/50 μl. The open bar represents the control without theinhibitors while the closed bar represents the adhesion to unstimulatedHUVEC monolayers. One standard deviation is indicated at the top of eachbar.

FIG. 6 shows the increase of lamp-1 molecules on the cell surface of SPcolonic carcinoma cells results in increased adhesion toE-selectin-expressing cells. Low metastatic colonic carcinoma SP cellsexpress a small amount of sialyl Le^(x) on cell surface lamp molecules(see the right cell). When the same cells were transfected with lamp-1expression vectors, the number of the cell surface lamp-1 molecules wasincreased. This increase was accompanied by an increased amount of cellsurface sialyl Le^(x) determinants and the increased efficiency ofadhesion to E-selectin-expressing cells (see the left cell).

DETAILED DESCRIPTION OF THE INVENTION

Lamp-1 and lamp-2 are the most abundant glycoproteins within thelysosomal membrane. Although the majority of lamp-1 and lamp-2 moleculesreside in lysosomes, some lamp-1 and lamp-2 are expressed on cellsurfaces (Lippincott-Schwartz et al., Cell 49:669-677 (1987); Mane etal., Arch. Biochem. Biophys. 268:360-378 (1989); Carlsson et al., Arch.Biochem. Biophys. 296:630-639 (1992) which are incorporated herein byreference), suggesting that those proteins can provide ligands forselectins. It has been shown that highly metastatic colonic carcinoma L4cells express more lamp-1 and lamp-2 on the cell surface than lowmetastatic SP cells (Saitoh et al., J. Biol. Chem. 267:5700-5711 (1992)incorporated herein by reference).

The lysosomal membrane glycoproteins, lamp-1 and lamp-2, are the majorcarriers of polylactosaminoglycans in various cells (Viitala et al.,Proc. Natl. Acad. Sci. USA 85:3743-3747 (1988); Carlsson et al., J.Biol. Chem. 263:18911-18919 (1988) both incorporated herein byreference), and as such are the major carriers forpoly-N-acetyllactosamines that are able to display sialyl Le^(x)termini. It has also been shown that lamp-1 is the major glycoproteincontaining GlcNAcβ1→6 Manα1→6Man branching in metastatic tumor cells asdetected by leukophytohemagglutin binding (Laferte et al., Biochem. J.259:569-576 (1989) incorporated herein by reference).

Polylactosaminoglycans are high molecular weight carbohydrates and aredistinguished from usual complex-type Asn-linked saccharides by havingside chains composed of endo-β-galactosidase susceptible(Galβ1→4GlcNAcβ1→3)_(n) repeats. The structures ofpolylactosaminoglycans are often characteristic to different cell typesand stages of differentiation. For example, the termini of humangranulocyte and monocyte polylactosaminoglycans are enriched in thesialyl Le^(x) moiety (NeuNAcα2→3Galβ1→4 (Fucα1→3) GlcNAc→R) whileerythrocyte polylactosaminoglycans termini are enriched inFucα1→2Galβ→4GlcNAc→R moieties, representing portions of the ABO bloodgroup antigen. It was discovered recently that the terminal structuresof poly-N-acetyllactosamines unique to granulocytes and monocytes serveas ligands for selectins present on endothelial cells and platelets. Ithas also been demonstrated that the isomer of sialyl Le^(x), sialylLe^(a) NeuNAcα-2→3Galβ1→3 (Fucα1→4) GlcNAc→R also serves as a ligand forE-selectin. Reports from several laboratories have shown that the levelof sialyl Le^(x) or sialyl Le^(a) is increased in tumor cells,particularly in carcinoma cells. It has also been demonstrated that sometumor cells adhere to endothelial cells by selectin-mediatedinteractions. These results suggest that tumor cells may adhere toendothelial cells at metastatic sites by the binding of E- or P-selectinto tumor cell surface carbohydrates.

In fact, it has been shown that highly metastatic colonic carcinoma celllines express more lamp-1 and lamp-2 on the cell surface than poorlymetastatic ones derived from a single human colon carcinoma (Saitoh etal., supra). It was also shown that the highly metastatic cell linescontain more poly-N-acetyllactosamine in carbohydrates attached to lampmolecules. These results suggest that tumor cells can modulate cellsurface display of selectin ligands by regulating levels of cell surfacelamp-1 and lamp-2 expression, and further suggest that upregulation ofsurface-localized lamp-1 and lamp-2 expression can therefore promote orfacilitate the metastatic process.

The present invention demonstrates that increased expression of lamp-1at the surface of colonic carcinoma cells leads to stronger adhesion toE-selectin expressing cells. Increased surface expression of lamp-1 wasachieved by over-expression of lamp-1 or by expression of mutated lamp-1that became a plasma membrane glycoprotein. Since low metastatic SPcells express only a small amount of lamp-1 on the cell surface, SPcells were chosen for increased expression of lamp-1 at the cell surfaceby gene transfer.

The results obtained in the Examples presented infra are consistent withthe fact that lamp molecules are the major carriers ofpoly-N-acetyllactosamines in nucleated cells. As shown previously(Mizoguchi et al., J. Biol. Chem. 259:11949-11957 (1984); Fukuda et al.,J. Biol. Chem. 260:1067-1082 (1985); Holmes et al., J. Biol. Chem.261:3737-3943 (1986) which are incorporated herein by reference),poly-N-acetyllactosamines appeared to be preferred substrates for α1→3fucosyltransferase and α2→3 sialyltransferase, thus efficientlyproviding sialyl Le^(x) termini. Since SP colonic cells apparentlycontain these glycosyltransferases, the amount of cell surface sialylLe^(x) is almost certainly directly related to the amount of sialylLe^(x) on the cell surface lamp-1 (see FIG. 6).

The present results are clearly consistent with the notion that sialylLe^(x) structures present in lamp-1 serve well as ligands for E-selectinon the cell surface. In a corroborative experiment, the present studyalso demonstrated that a soluble lamp-1 can inhibit E-selectin mediatedadhesion and such inhibition can be obtained only when the solublelamp-1 was prepared from cells expressing sialyl Le^(x) structures. Theresults demonstrate that lamp-1 can be an efficient inhibitor forE-selectin-mediated adhesion.

It was shown that sialyl Le^(x) terminal structures can be present inglycolipids as well (Fukushima et al., Cancer Res. 44:5279-5285 (1984)incorporated herein by reference). Such glycolipids are particularlyenriched in colonic carcinoma cells and it is plausible to assume thatSP cells also contain such glycolipids. In the present study, theincreased amount of lamp-1 significantly increased the adhesion of SPcells to endothelial cells and such adhesion can be inhibitedefficiently by soluble lamp1- that contains sialyl Le^(x) structures.These results suggest that sialyl Le^(x) structures present onglycoproteins may be better ligands than those on glycolipids. It ispossible that glycans attached to proteins may extend more, enablingselectin molecules to bind to the presented ligand (see Moore et al., J.Cell Biol. 118:445-456 (1992) incorporated herein by reference).

It was recently shown that the nonspecific cross-reacting antigen CD66can inhibit E-selectin mediated adhesion (Kuijpers et al., J. Cell Biol.118:457-466 (1992) incorporated herein by reference). CD66 is a memberof the immunoglobulin superfamily and was shown to contain sialyl Le^(x)termini when it was purified from meconium (Yamashita et al., J. Biol.Chem. 264:17873-17881 (1989) incorporated herein by reference). Althoughit is not clear if CD66 is the major presenter of sialyl Le^(x) onneutrophils, the results are consistent with the present results,showing that a sialyl Le^(x) -containing glycoprotein can inhibitE-selectin mediated adhesion. On the other hand, it was reportedrecently that P-selectin binds preferentially to a glycoprotein withMr.˜120,000 (Moore, Supra). This molecule is different from lampmolecules or leukosialin, although both lamp (Saitoh et al., supra) andleukosialin (Maemura et al., J. Biol. Chem. 267:24379-24386 (1992)incorporated herein by reference) were found to contain sialyl Le^(x)structures. It was shown that L-selectin on neutrophils may presentsialyl Le^(x) to E-selectin (Picker et al., Cell 66:921-933 (1991)incorporated herein by reference). On the other hand, L-selectin wasfound to preferentially bind to two glycoproteins on endothelial cells.Most recently a report demonstrated that one of those glycoproteins is aleukosialin-like glycoprotein containing a multiple number of O-glycans(Lasky et al., Cell 69:927-938 (1992) incorporated herein by reference).It is thus likely that glycans presented on this glycoprotein, termedGlycam, provide a better ligand(s) for L-selectin. It thus seemsreasonable to assume that selectin ligands are preferentially presentedto selectins by a limited number of glycoproteins. Nevertheless, thepresent study strongly suggests that lamp-1 may function to presentsialyl Le^(x) and also sialyl Le^(a) on tumor cells, and most likelyalso on other cell types. Furthermore, our present study demonstratedthat lamp-1 can be used as an efficient inhibitor in E-selectin mediatedadhesion.

These results also strongly suggest that tumor cells utilizeselectin-carbohydrate interaction when tumor cells adhere at metastaticsites. In fact, tumor cells, in particular carcinoma cells, have beenshown to be enriched with sialyl Le^(x) and sialyl Le^(a) structures(Magnani et al., J. Biol. Chem. 257:14365-14369 (1982); Fukushima,supra; Kim et al., Cancer Res. 48:475-482 (1988) incorporated herein byreference), which are ligands for E- and P-selectins (Lowe et al., Cell63:475-484 (1990); Phillips et al., Science 250:1130-1132 (1990); Walzet al., Science 250:1132-1135 (1990); Larsen et al., Cell 63:467-474(1990); Berg et al., J. Biol. Chem. 266:14869-14972 (1991); Polley etal., Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991) incorporated hereinby reference). It has also been shown that some tumor cells aggregatewith platelets (Nicolson, G. L., Curr. Opinion Cell Biol. 1:1009-1019(1989) incorporated herein by reference) that presumably express E- andP-selectin. Such aggregated cells then could be trapped in capillarytubes, which could then trigger the activation of endothelial cellsleading to the expression of E-selectin. It is possible that theseevents result in the lodging of tumor cells in capillary beds atjunctions between endothelial cells (Nicolson, supra). In the case ofneutrophil adhesion during inflammation, it has been shown that suchE-selectins mediated adhesion leads to stronger adhesion to endothelialcells through integrins and counter receptor interaction (Springer, T.A., Nature (Lond.) 346:425-434 (1990) incorporated herein by reference).Once such interaction is established, neutrophils cross the boundarybetween endothelial cells and then establish extravasation. It wasdemonstrated that neutrophil extravasation can be inhibited byinhibition of the first step, rolling effect, with anti-E-selectinantibody (Lawrence et al., Cell 65:859-873 (1991); Ley et al., Blood77:2553-2555 (1991) incorporated herein by reference). Recently, it wasshown that E-selectin mediates acute lung inflammation induced bydeposition of IgG immune-complexes (Mulligan et al., J. Clin. Invest.88:1396-1406 (1991) incorporated herein by reference). Most recentstudies demonstrated that such inflammation can be inhibited byadministration of sialyl Le^(x) -glycopeptides or oligosaccharides. Thepresent study strongly suggests that sialyl Le^(x) positive, solublelamp-1 can be an efficient inhibitor in such inflammatory processes. Theestablishment of tumor metastasis is reminiscent of this process and itis not unreasonable to assume that tumor cells may utilize the samemechanism during metastatic spread. The present invention, thus,provides means for binding selectin receptors on platelets andendothelial cells that have been activated by an immune response,thereby inhibiting or preventing binding to the selectin receptor of thenative membrane-bound lamp polypeptide. The pathological conditionsintended to be affected comprise, but are not limited to carcinoma cellsthat express sialyl Le^(x) and/or sialyl Le^(a) antigenic determinants,for example colon, breast, stomach, pancreatic and lung carcinoma cells.The pathological conditions intended also include leukemic cells thatexpress sialyl Le^(x) and/or sialyl Le^(a) determinants as a means toescape from blood vessels into the body fluid, for example acute andchronic myelogenous leukemia cells. Other pathological conditions arethose involving adhesion of circulating leukocytes to the vascularendothelium during inflammatory diseases, for example,ischemia-reperfusion injury that often occurs as a concomitant ofmyocardial infarction and stroke and inflammatory conditions of thelung.

Accordingly, the present invention provides soluble lamp-derivedpolypeptides having sialylated carbohydrate antigens that bind selectinreceptors on the cell surface. The nucleic acid sequence encoding thesoluble lamp-1 polypeptide is included within the sequence set forth inTable I (SEQ. ID NOS: 1,2,17,18)(from about nucleic acid number 180 toabout nucleic acid number 1330). The nucleic acid sequence encoding the,soluble lamp-2 polypeptide is included within the sequence set forth inTable II (SEQ. ID NOS: 3-11, 16) (from about nucleic acid number 119 toabout nucleic acid number 1568). Depending upon the cell type, purifiedmature lamp polypeptide has a molecular mass between 90-120 kD, whereasthe soluble form has a molecular mass between 70-100 kD.

    TABLE I      -      GAATTCGGGCGGGCTTCTTCGCTGCCGACGTACGACGAGTGGCCGGGCTCTTGCGTCTGGTAACGCGCTGTCT    C     03                                                                              G    C     90      ##STR1##      ##STR2##      ##STR3##      ##STR4##      ##STR5##      ##STR6##      ##STR7##      ##STR8##      ##STR9##      ##STR10##      ##STR11##      ##STR12##      ##STR13##      ##STR14##      ##STR15##                                                                              T    A     600      ##STR16##      ##STR17##      ##STR18##      CATATCATTGAGTTTAGGGTTCTGGTGTTTGGTTTCTTCATTCTTTACTGCACTCAGATTTAAGCCTTACAAA    G     020                                                                              T    G     125                                                                              T    T     230                                                                              T    T     335      ##STR19##                                                                              A    A     455

    TABLE II      -      ##STR20##      ##STR21##      ##STR22##      ##STR23##      ##STR24##      ##STR25##      ##STR26##      ##STR27##      ##STR28##      ##STR29##      ##STR30##      ##STR31##      ##STR32##      ##STR33##      ##STR34##      ##STR35##      ##STR36##      TTGGTTTTCAGTTGAATGAAGTAGAG

As used herein, the term "purified" means that the molecule or compoundis substantially free of contaminants normally associated with a nativeor natural environment. For example, the mature membrane-associatedlamp-1 and lamp-2 polypeptides can be isolated from various methods wellknown to a person of skill in the art. The methods available for thepurification of membrane proteins include precipitation, gel filtration,ion-exchange, reverse-phase and affinity chromatography. Otherwell-known methods are described in Deutscher et al., Guide to ProteinPurification: Methods in Enzymology Vol. 182, (Academic Press 1990),which is incorporated herein by reference. Alternatively, the purifiedpolypeptides of the present invention can also be obtained by well-knownrecombinant methods as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring HarborLaboratory 1989), also incorporated herein by reference. An example ofthe means for preparing soluble lamp-derived polypeptide is to expressnucleic acid encoding the soluble lamp in a suitable host cell, such asa bacterial, yeast or mammalian cell, using methods well known in theart, and recovering the expressed soluble protein, again using methodswell known in the art. The soluble polypeptide and biologically activefragments thereof can also be produced by chemical synthesis. Syntheticpolypeptides can be produced using Applied Biosystems, Inc. (FosterCity, Calif.) Model 430A or 431A automatic polypeptide synthesized andchemistry provided by the manufacturer. The soluble polypeptides canalso be isolated directly from cells that have been transformed withexpression vectors, described below in more detail.

As used herein, "lamp-derived polypeptide" means a polypeptide havingthe amino acid sequence substantially the same as the amino acidsequence shown in Table I for lamp-1 or Table II for lamp-2, or activefragments thereof. As used herein the term "soluble lamp-derivedpolypeptide" refers to a soluble, biologically active fragment of thehuman lamp-1 or human lamp-2 polypeptide expressed by the extracellulardomain of its respective nucleic acid. Further, "soluble polypeptide"refers to a non-naturally occurring cleaved polypeptide that functionsas a secreted molecule and retains the ability to bind to the ligandsrecognized by its membrane counterpart, for example, cell surfaceselectin receptors. As used herein, an "active fragment" or"biologically active fragment" refers to any portion of the lamppolypeptide shown in Table I (SEQ ID NO: 2) or Table II (SEQ ID NO: 16)that binds to E- and/or P-selectin receptors. Methods to determine lampbinding to selectin receptors are well known to those of skill in theart, for example, as set forth below.

The invention also encompasses nucleic acid molecules which differ fromthat of the nucleic acid molecules shown in Table I (SEQ ID NO: 1) orTable II, (SEQ ID NOS: 3-11) but which produce the same phenotypiceffect. These altered, but phenotypically equivalent nucleic acidmolecules are referred to as "equivalent nucleic acids". This inventionalso encompasses nucleic acid molecules characterized by changes innon-coding regions that do not alter the phenotype of the polypeptidesproduced therefrom when compared to the nucleic acid molecule describedhereinabove. This invention provides a nucleic acid molecule encodingsoluble lamp-derived polypeptide wherein said nucleic acid molecule hasbeen mutated such that the lysosomal targeting signal encoded therebyhas been rendered non-functional. Methods to mutate nucleic acidmolecules are well known in the art. An example of such method issite-directed mutagenesis. This invention additionally provides nucleicacid molecules which hybridize to the nucleic acid molecules of thesubject invention. As used herein, the term "nucleic acid" encompassesRNA as well as single and double-stranded DNA and cDNA. As used herein,the term "polypeptide" encompasses any naturally occurring allelicvariant thereof as well as recombinant forms.

This invention provides an isolated nucleic acid molecule encoding ahuman soluble lamp-derived polypeptide. As used herein, the term"isolated nucleic acid molecule" means a nucleic acid molecule that isin a form that does not occur in nature. Once means of isolating a humanlamp-1 or human lamp-2 nucleic acid is to probe a human cDNA expressionlibrary with a natural or artificially designed antibody to human lamp-1or human lamp-2, using methods well known in the art (see Gougos et al.,J. Biol. Chem. 265:8361 (1990) which is incorporated herein byreference). DNA and cDNA molecules which encode human lamp polypeptidescan be used to obtain complementary genomic DNA, cDNA or RNA from humanor other mammalian sources.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes human lamp-1 or an mRNA molecule which encodes humanlamp-2 so as to prevent translation of the mRNA molecule. The antisenseoligonucleotide can have a sequence capable of binding specifically withany sequences of the cDNA molecule, the sequence of which is shown inTable I or Table II. As used herein, the phrase "binding specifically"encompasses the ability of a nucleic acid sequence to recognize anucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs. A particular example of an antisense oligonucleotide is anantisense oligonucleotide comprising chemical analogues of nucleotides.

This invention also provides a pharmaceutical composition comprising anamount of the oligonucleotide described above effective to reduceexpression of a human lamp-1 or human lamp-2 polypeptide by passingthrough a cell membrane and binding specifically with mRNA encoding ahuman lamp-1 or human lamp-2 polypeptide in the cell so as to preventits translation and a pharmaceutically acceptable hydrophobic carriercapable of passing through a cell membrane. The pharmaceuticallyacceptable hydrophobic carrier capable of passing through cell membranesmay also comprise a structure which binds to a receptor specific for aselected cell type and is thereby taken up by cells of the selected celltype. The structure may be part of a protein known to bind to acell-type specific receptor, for example, an insulin molecule whichwould target pancreatic cells. As used herein, the term"pharmaceutically acceptable carrier" encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water and emulsions such as an oil/water or water/oil emulsion, andvarious types of wetting agents.

Antisense oligonucleotide drugs inhibit translation of mRNA encodingthese polypeptides. Synthetic oligonucleotides, or other antisensechemical structures are designed to bind to mRNA encoding the lamppolypeptides and inhibit translation of mRNA and are useful as drugs toinhibit expression of lamp-1 and lamp-2 polypeptide genes in patients.This invention provides a means to therapeutically alter levels ofexpression of human lamp polypeptides by the use of a syntheticantisense oligonucleotide drug (hereinafter SAOD) which inhibitstranslation of mRNA encoding these polypeptides. Syntheticoligonucleotides, or other antisense chemical structures designed torecognize and selectively bind to mRNA, are constructed to becomplementary to portions of the nucleotide sequences shown in Table Ior Table II of DNA, RNA or chemically modified, artificial nucleicacids. The SAOD is designed to be stable in the blood stream foradministration to patients by injection, or in laboratory cell cultureconditions. The SAOD is designed to be capable of passing through thecell membrane in order to enter the cytoplasm of the cell by virtue ofphysical and chemical properties of the SAOD which render it capable ofpassing through cell membranes, for example, by designing small,hydrophobic SAOD chemical structures, or by virtue of specific transportsystems in the cell which recognize and transport the SAOD into thecell. In addition, the SAOD can be designed for administration only tocertain selected cell populations by targeting the SAOD to be recognizedby specific cellular uptake mechanisms which bind and take up the SAODonly within select cell populations. For example, the SAOD may bedesigned to bind to a receptor found only in a certain cell type, asdiscussed supra. The SAOD is also designed to recognize and selectivelybind to the target mRNA sequence, which may correspond to a sequencecontained within the sequence shown in Table I or Table II. The SAOD isdesigned to inactivate the target mRNA sequence by either binding to thetarget mRNA and inducing degradation of the mRNA by, for example, RNaseI digestion, or inhibiting translation of the mRNA target by interferingwith the binding of translation-regulating factors or ribosomes, orinclusion of other chemical structures, such as ribozyme sequences orreactive chemical groups which either degrade or chemically modify thetarget mRNA. SAOD drugs have been shown to be capable of such propertieswhen directed against mRNA targets (see Cohen et al., TIPS, 10:435(1989) and Weintraub, Sci. American, January (1990), pp.40; bothincorporated herein by reference). An SAOD serves as an effectivetherapeutic agent if it is designed to be administered in vivo or exvivo. In this manner, an SAOD serves as a therapy to reduce lamppolypeptide expression in particular target cells of a patient, in aclinical condition which may benefit from reduced expression of lamppolypeptides, inflammatory responses and tumor cell adhesion reactionsthat lead to metastasis.

The invention further provides an isolated nucleic acid moleculeoperatively linked to a promoter of RNA transcription, as well as otherregulatory sequences. As used herein, the term "operatively linked"means positioned in such a manner that the promoter will direct thetranscription of RNA off of the nucleic acid molecule. Examples of suchpromoters are SP6, T4 and T7. Vectors which contain both a promoter anda cloning site into which an inserted nucleic acid is operatively linkedto that promoter are well known in the art. Preferably, these vectorsare capable of transcribing RNA in vitro or in vivo. Examples of suchvectors are the pGEM series (Promega Biotech, Madison, WI).

This invention provides a vector comprising an isolated nucleic acidmolecule such as DNA, cDNA or RNA encoding a soluble lamp-derivedpolypeptide. Examples of such vectors are viruses, such asbacteriophages, baculoviruses and retroviruses; cosmids, plasmids andother recombination vectors. Nucleic acid molecules are inserted intovector genomes by methods well known in the art. For example, insert andvector DNA can both or individually be exposed to restriction enzymes tocreate complementary ends on both molecules that base pair with eachother and which are then joined together with a ligase. Alternatively,synthetic nucleic acid linkers can be ligated to the insert DNA thatcorrespond to a restriction site in the vector. The vector is thendigested with the respective restriction enzyme and the respectivenucleic acid may then be inserted. Additionally, an oligonucleotidecontaining a termination codon and an appropriate restriction site canbe ligated into a vector containing, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; versatilemultiple cloning sites; and T7 and SP6 promoters for in vitrotranscription of sense and anti-sense RNA. Other means are available.

Also provided are vectors comprising a DNA molecule encoding a humansoluble lamp-derived polypeptide, adapted for expression in a bacterialcell, a yeast cell, a mammalian cell and other animal cells. The vectorsadditionally comprise the regulatory elements necessary for expressionof the DNA in the bacterial, yeast, mammalian or animal cells so locatedrelative to the DNA encoding soluble lamp-derived polypeptide as topermit expression thereof. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Maniatis et al., supra 1989). Similarly a eucaryotic expressionvector includes a heterologous or homologous promoter for RNA polymeraseII, a downstream polyadenylation signal, the start codon AUG, and atermination codon for detachment of the ribosome. Such vectors can beobtained commercially or assembled by methods well known in the art, forexample, the methods described above for constructing vectors ingeneral. Expression vectors are useful to produce cells that express thepolypeptide.

This invention provides a mammalian cell containing a cDNA moleculeencoding a human soluble lamp-derived polypeptide. An example is amammalian cell comprising a plasmid adapted for expression in amammalian cell. The plasmid has a cDNA molecule encoding a solublelamp-derived polypeptide and the regulatory elements necessary forexpression of the polypeptide. Various mammalian cells may be utilizedas hosts, including, for example, mouse fibroblast cell NIH3T3, CHOcells, HeLa cells, Ltk- cells, etc. Expression plasmids such as thosedescribed supra can be used to transfect mammalian cells by methods wellknown in the art such as calcium phosphate precipitation, DEAE-dextran,electroporation, microinjection or lipofection.

This invention provides a pharmaceutical composition containing apharmaceutical carrier and any of a purified, soluble polypeptide, anactive fragment thereof, or a purified, mature protein and activefragments thereof, alone or in combination with each other. Thesepolypeptides or proteins can be recombinantly derived, chemicallysynthesized or purified from native sources. As used herein, theterm"pharmaceutically acceptable carrier" encompasses any of thestandard pharmaceutical carriers, such as phosphate buffered salinesolution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents.

Also provided are antibodies having specific reactivity with thelamp-derived polypeptides of the subject invention. Active fragments ofantibodies are encompassed within the definition of "antibody". Theantibodies of the invention can be produced by any method known in theart. For example, polyclonal and monoclonal antibodies can be producedby methods well known in the art, as described, for example, in Harlowand Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory1988), which is incorporated herein by reference. The polypeptide,particularly soluble lamp-derived polypeptide of the present invention,can be used as the immunogen in generating such antibodies. Alteredantibodies such as chimeric, humanized, CDR-grafted or bifunctionalantibodies can also be produced by methods well known in the art. Suchantibodies can also be produced by hybridoma, chemical synthesis orrecombinant methods described, for example, in Sambrook et al., supra,incorporated herein by reference. The antibodies can be used fordetermining the presence or purification of the soluble lamp-derivedpolypeptides of the present invention. With respect to the detection ofsuch polypeptides, the antibodies can be used for in vitro diagnostic orin vivo imaging methods.

Immunological procedures useful for in vitro detection of the targetsoluble lamp-derived polypeptide in a sample include immunoassays thatemploy a detectable antibody. Such immunoassays include, for example,ELISA, Pandex microfluorimetric assay, agglutination assays, flowcytometry, serum diagnostic assays and immunohistochemical stainingprocedures which are well known in the art. An antibody can be madedetectable by various means well known in the art. For example, adetectable marker can be directly or indirectly attached to theantibody. Useful markers include, for example, radionuclides, enzymes,fluorogens, chromogens and chemiluminescent labels.

This invention provides a method of modifying a biological functionmediated by the regulatory activity of a selectin receptor whichcomprises contacting a suitable sample containing a selectin receptorwith an effective amount of a biologically active lamp-derivedpolypeptide or a pharmaceutical composition described above. As usedherein "an effective amount" refers to an amount of the polypeptidesufficient to bind to a selectin receptor and thereby prevent or inhibitits regulatory activity. This method is especially useful for modifyingthe regulatory activity of E-selectin or P-selectin. Examples ofregulatory activities include, but are not limited to mediation ofcellular adhesion to endothelial cells and platelets.

An effective amount is any amount that is effective to modify thebiological function mediated by the regulatory activity of E- and/orP-selectin receptors. The method can be practiced in vitro, ex vivo orin vivo. If the method is practiced in vitro, contacting is effected byincubating the sample with a polypeptide, a protein or a pharmaceuticalcomposition described above. The ex vivo method is similar but includesthe additional step of reintroducing the treated sample into thesubject.

However, in a preferred embodiment the contacting is effected in vitroby administering a polypeptide, a protein or a pharmaceuticalcomposition, as described above to a subject, e.g., a mammal or a human.

Methods of administration are well known to those of skill in the artand include, but are not limited to, administration orally,intravenously or parenterally. Administration will be in such a dosagethat the regulatory activity is effectively modified. Administration canbe effected continuously or intermittently such that this amount iseffective for its intended purpose.

This invention also provides a method of alleviating a pathologiccondition caused by a selectin-mediated activity comprising contactingthe selectin receptor with any of a purified soluble lamp-derivedpolypeptide, an active fragment thereof, a lamp-derived polypeptide oran active fragment thereof. The selectin receptor is bound with saidpolypeptide to treat the pathologic condition mediated by selectinreceptor activity. As used herein "pathologic conditions" refers to anypathology arising from selectin receptor induced regulatory activity.For example, tumor cell adhesion to endothelium and leukocyte adhesionto inflammatory sites are selectin receptor mediated events.

In a preferred embodiment, the method is practiced by administering to asubject, an effective amount of a purified lamp-derived protein or apurified soluble lamp-derived polypeptide or a biologically activefragment thereof, or the pharmaceutical composition described above.Methods of administration are outlined supra.

This invention also provides a method of detecting the presence ofselectin receptors on endothelial cells comprising contacting a sampleof endothelial cells with a lamp-derived polypeptide, detecting bindingof the lamp polypeptide to the selectin receptor, wherein said bindingindicates that the endothelial cell is in an activated state (seeExample IX infra).

It is understood that modifications which do not substantially affectthe activity of the various molecules of this invention are alsoincluded within the definition of said molecules.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Plasmid Preparation and Vector Construction

A cDNA encoding the lamp-1 molecule, designated L1-15/202 (Williams etal., J. Cell Biol. 111:955-966 (1990) incorporated herein by reference),was inserted into Bluescript (Stratagene, La Jolla, Calif.), resultingin pBL1-15/202. This cDNA contains the full-length coding sequence ofhuman lamp-1 and a truncated 3'-flanking sequence. After XhoI and BamHIdigestion, the cDNA insert was cloned into a pSV vector, resulting inpSVlamp-1. The pSV vector is a derivative of pJC119 (Guan et al., Cell37:779-787 (1984) incorporated herein by reference). The mutant cDNA inwhich the cytoplasmic tyrosine is replaced with histidine was made asdescribed (Williams et al., supra), and constructed in the same vector,resulting in pSVlamp-1^(H).

The wild-type and mutant lamp-1 cDNAs were cloned in parallel intopcDL-SRα-478. The pcDL-SRα vector contains the SV40 promoter and theHTLV-1 LTR (Takebe et al., Mol. Cell. Biol. 8:466:472 (1988)incorporated herein by reference). pcDL-SRα-478 was derived frompcDL-SRα-296 to generate an EcoRI cloning site. cDNAs encoding wild-typelamp-1 and its mutant were excised from the pSV vectors described aboveby XhoI and BamHI and blunt-ended with the Klenow fragment of DNApolymerase I. pcDL-SRα-478 vector was digested by EcoRI, and EcoRI siteswere also blunt-ended with the Klenow fragment of DNA polymerase I. Eachblunt-ended cDNA was then ligated between the blunt EcoRI sites ofpcDL-SRα-478. Clones containing a single lamp-1 cDNA, or mutant lamp-1cDNA, in the proper orientation, were designated pSRαlamp-1 andpSRαlamp-1^(H), respectively.

Plasmid pcDNA1-Fuc-TIII was constructed by first isolating fromPCDM7-α(1,3/1,4)FT (Kukowska-Latallo et al. Genes Develop. 4:1288-1303(1990); Weston et al. J. Biol. Chem. 267:24575-24584 (1992) incorporatedherein by reference) a 2.2 kb XhoI fragment corresponding to theFuc-TIII cDNA. This fragment was then cloned into the sense orientationinto the unique XhoI site in plasmid pcDNAI (Invitrogen, San Diego,Calif.).

EXAMPLE II Amplification of E-selectin Sequences

Human E-selection cDNA sequence was amplified by polymerase chainreaction (PCR) (Higuchi et al., Nucl. Acids Res. 16:7351-7367 (1988)incorporated herein by reference) using a human endothelial cDNA library(Staunton et al., Cell 52:925-933 (1988) incorporated herein byreference) as a template. The cDNA library was constructed from mRNA ofactivated human endothelial cells (Staunton et al., supra). The5'-,primer sequence was 5'-TCAAGTACTCTTGAAGTCATGATTGCTTCA-3' (SEQ ID NO:12) and corresponds to -9 to +12 nucleotides with respect to thetranslation initiation codon with a ScaI restriction site at the 5'-end.The 3'-primer sequence was 5'-TGAAGTACTAACTTAAAGGATGTAAGAAGGCTT-3' (SEQID NO 13). This sequence corresponds in anti-sense to -18 to +6 bp withrespect to the stop codon with the ScaI restriction site at the 5' end.Amplification of cDNA was achieved after 40 cycles under the followingconditions: denaturation for 1 minute at 94° C., annealing for 2 minutesat 55° C., and polymerization for 3 minutes at 72° C. The amplified DNAwas cut with ScaI and cloned into the EcoRV site of Bluescript/ks. ThecDNA insert was then excised by digestion with ScaI and KpnI.pcDL-SRα-478 was first digested with EcoRI and the EcoRI site wasblunt-ended by the Klenow fragment of DNA polymerase I. The blunt-endedplasmid was then digested with KpnI, which is situated 3' to the EcoRIsite. The SmaI-KpnI fragment representing the E-selectin cDNA was clonedbetween the blunt EcoRI-KpnI ends of pcDL-SRα-478 to yieldpSRα-E-selectin.

EXAMPLE III Amplification of Lamp Sequences

A cDNA encoding soluble lamp-1 was amplified by PCR using the pSV lamp-1as a template. The 5'-primer sequence was5'-TTTGAATTCCTCGCGCCATGGCGCC-3' (SEQ ID NO: 14). This corresponds to -8to +8 bp relative to the initiation codon plus an EcoRI restriction siteand TTT at 5'-end. The 3'-primer sequence is5'-AAAGGTACCTAGCTGTTCTCGTCCAGCAG-3' (SEQ ID NO: 15. This sequencecontains the lamp-1 sequence in anti-sense from codons 348 to 353, afterwhich a stop codon is introduced. The sequence also contains AAA plus aKpnI site. After amplification under the same conditions describedabove, the DNA was cut with EcoRI and KpnI and then cloned into theEcoRI/KpnI sites of pcDL-SRα-478, to yield pSRαs-lamp-1.

EXAMPLE IV Establishment of SP Colonic Cells Expressing Various Amountsof Lamp-1 on the Cell Surface

The isolation and characterization of the poorly metastatic human coloncarcinoma line KM12-SP (hereinafter SP) has been previously described(Saitoh et al., supra; Morikawa et al., Cancer Res. 48:1943-1948 (1988)incorporated herein by reference). This cell line expresses less lamp-1on the cell surface than its highly metastatic counterpart, L4 (Saitohet al., supra). SP colonic cells are poorly metastatic in nude mouseexperiments and express only 3% of the total lamp-1 on the cell surface(Saitoh et al., supra).

In order to establish SP cells that express an increased amount of cellsurface lamp-1, SP colonic cells were transfected with vectors thatexpress wild-type, membrane-tethered lamp-1 (pSVlamp-1 and pSRαlamp-1).The cells were co-transfected with pSV₂ neo. The ratio of plasmidsharboring lamp-1 cDNAs and pSV₂ neo was 10:1. After transfection, thecells were selected with G418 (1 mg/ml), in DME containing 10% fetalcalf serum, sodium pyruvate, MEM vitamin solution, non-essential aminoacids, and antibiotics. After culturing for 10 days in the presence ofG418, clonal cell lines were obtained by limiting dilution and differentclones were examined by immunofluorescence for cell surface expressionof lamp-1.

Similarly, in a second set of experiments, increased lamp-1 cell surfaceexpression was sought by expressing a mutant lamp-1 in which acytoplasmic tyrosine residue critical for lysosomal targeting (Williamset al., supra) had been changed to a histidine residue (vectorspSVlamp-1^(H) and pSRαlamp-1^(H)). The resulting mutant lamp-1 molecule,in contrast to its wild-type counterpart, does not sort to the lysosome,and therefore accumulates preferentially at the cell surface via itsdefault biosynthetic pathway.

Several SP cell lines were derived from transfections with each of thefour lamp-1 vectors. FIG. 1 illustrates a flow cytometry analysis ofcell surface lamp-1 expression in representative clones containing thesedifferent vectors. These cell lines each express roughly at leasttwo-fold more cell surface lamp-1 (mean fluorescent intensities ˜20,˜35, ˜40, ˜60) than does the parental SP cell line (mean fluorescentintensity=˜13). Cells transfected with pSRαlamp-1 express more cellsurface lamp-1 (FIG. 1B, mean fluorescence intensity=˜35) than do cellstransfected with pSVlamp-1 (FIG. 1A, mean fluorescent intensity=˜20),and cells expressing the mutant lamp-1 express more surface-localizedlamp-1 than do cells expressing the wild-type lamp-1 (FIG. 1, comparepanels C and D, mean fluorescence intensities=˜40 and ˜60, respectively,versus panel A and B, respective mean fluorescence intensities of ˜20and ˜35).

EXAMPLE V Establishment of CHO Cell Lines Expressing E-Selectin

CHO cells were co-transfected with pSRαE-selectin and pSV₂ dhfr in a10:1 molar ratio, using the lipofectin procedure. After thetransfection, cells were cultured in α-MEM without nucleotides for 14days. The cells were then propagated with increasing concentration ofmethotrexate (Sigma, St. Louis, Mo.) final concentration of 0.5 μM andcloned cell lines were obtained in 24-well tissue culture plates. Eachclone was tested for HL-60 cell binding as a screen for cell surfaceE-selectin expression. Clones that efficiently bound HL-60 cells weresubsequently tested by immunofluorescence using an anti-E-selectinantibody to confirm E-selectin expression.

EXAMPLE VI Expression of Soluble Lamp-1 in CHO Cells

In order to produce a soluble lamp-1 molecule that displays sialylLe^(x) determinants, CHO cells were first co-transfected by thelipofectin procedure (Bierhuizen et al., Proc. Natl. Acad. Sci. USA84:9326-9330 (1992) incorporated herein by reference) withpcDNA1-Fuc-TIII and pHyg (Sugden et al., Molec. Cell. Biol. 5:410-413(1985) incorporated herein by reference) in a 10:1 molar ratio. Thetransfected cells were selected in the presence of 500 μg/ml ofhygromycin (Sigma, St. Louis, Mo.) and the cloned in a 24-well tissueculture plate. Each cell line was assessed for the expression of sialylLe^(x) by immunofluorescence. Immunofluorescence staining was carriedout using a mouse monoclonal anti-sialyl Le^(x) antibody, CSLEX(Fukushima et al., supra) (purchased from UCLA tissue culturelaboratory), followed by staining with rhodamine-conjugated goatanti-mouse IgM, using procedures described previously (Williams et al.,supra).

A clonal cell line stably expressing sialyl Le^(x) molecules was thenco-transfected with pSRα s-lamp-1 and pSV₂ dhfr, and the transfectedcells were selected in α-MEM without nucleotides. After culturing underthese conditions for 14 days, the cells were propagated for geneamplification by methotrexate as described above. Expression of solublelamp-1 was determined by immunoblotting of the conditioned medium. Theconditioned medium from each well was concentrated (Centricon 30, AmiconInc., Beverly, Mass.), and the concentrated medium was applied to anitrocellulose membrane. After blocking with 5% milk in PBS, themembrane was incubated at room temperature for 1 hour with rabbitanti-lamp-1 antibody diluted in 20 mM Tris-HCl, pH 7.5 containing 1% BSAand 0.5M NaCl (buffer A). The membrane was then washed for 5 minutes atroom temperature with 20 mM Tris-HCl, pH 7.5 containing 0.5M NaCl and0.05% Tween-20 twice and with the same buffer without Tween-20, and thenincubated with alkaline phosphatase-conjugated goat anti-rabbit antibodyin buffer A. The blot was then washed with the same buffer, andincubated with alkaline phosphatase substrate(5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium in 10 mMTris-HCl, 2 mM MgCl₂, pH 9.0) using procedures previously described(Blake et al., Anal. Biochem. 136:175-179 (1984) incorporated herein byreference). One cell line that produced an abundant amount of solublelamp-1, as determined with this procedure, was chosen for further study.

EXAMPLE VII Purification of Soluble Lamp-1 from the Conditioned Mediumof CHO Cells

The CHO cell line expressing soluble lamp-1 was cultured in α-MEMcontaining 0.5 μM methotrexate, and the medium was replaced withOpti-MEM (BRL, Bethesda, Md.) after the cells reached confluency. Afterculturing for 3 days, the conditioned medium (260 ml) was collected andapplied to a column (1.2×2.5 cm) of wheat germ agglutinin-Agarose (E-YLaboratories, San Mateo, Calif.). The column was equilibrated with 10 mMpotassium phosphate buffer, pH 7.4 containing 0.14M NaCl and eluted with100 mM GlcNAc in the same buffer. The eluate was dialyzed against 50 mMpotassium phosphate buffer, pH 7.0, containing 1 mM EDTA and the samplewas applied to a column (2 ml) of DEAE-Sephadex (Sigma, St. Louis, Mo.)equilibrated with the same buffer. The column was eluted with the samebuffer containing 0.1M NaCl without EDTA. The eluted sample was thendialyzed against PBS, and tested in adhesion assays.

EXAMPLE VIII Flow Cytometry Analysis

SP cells expressing various amounts of cell surface lamp-1 were stainedwith the mouse IgG anti-human-lamp-1 antibody, BB6 (Carlsson et al., J.Biol. Chem. 264(34):20526-20531 (1989) incorporated herein byreference); ascites diluted 1:500. Cells were then stained withfluorescein-conjugated goat anti-mouse IgG (40 μg/ml)(Sigma, St. LouisMo.) and subjected to analysis by flow cytometry on a FACScan (BectonDickinson, Mountain View, Calif.). Cell staining was measured inarbitrary units as the log of fluorescent intensity and displayed on afour decade scale.

EXAMPLE IX Colonic Carcinoma Cells Adhere to IL-1β Treated EndotheliaThrough E-selectin Binding to Sialyl Le^(x) Structures

The four lamp-1 transfected SP cell lines (SP-pSVlamp-1, SP-pSRαlamp-1,SP-pSVlamp-1^(H), or SP-pSRαlamp-1^(H)), and the control SP cell linewere then subjected to adhesion assays to determine their relativeabilities to exhibit E-selectin-dependent adhesive properties.

Adhesion of SP cells to human umbilical vein endothelial cells,hereinafter HUVEC, (Clonetics, San Diego, Calif.) was carried out asdescribed previously (Phillips et al., supra) with a slightmodification. Briefly, SP cells were metabolically labeled with [³⁵S]-methionine (100 μCi/ml, ICN) in methionine-free DME for 2 hours asdescribed previously (Lee et al., J. Biol. Chem. 265:20476-20487 (1990)incorporated herein by reference). The [³⁵ S]-methionine labeled SPcells were harvested in the cell dissociation solution (Specialty Media,Lavellette, N.J.) and washed twice with DME before assay of the bindingto HUVEC. HUVEC monolayers cultured in 96-well tissue culture plates,were activated with 5 unit/ml of IL-1β (Boehringer-Mannheim,Indianapolis, Ind.) for 4 hours and then washed with DME containing 5%fetal calf serum. Control non-activated HUVEC monolayers were preparedidentically (without II-1β), and used in parallel for adhesion assays.

Approximately 1×10⁵ of ³⁵ S-labeled SP cells were added to the HUVECmonolayers in 0.1 ml of DME containing 5% fetal calf serum. Afterincubation at 37° C. for 15 minutes, the cells were washed with the samesolution three times. Adherent cells remaining after washing weredissolved in 0.1 ml of cell dissolution solution. The solutioncontaining the solubilized cells was added to 2 ml of Aquamixscintillation cocktail, and radioactivity was determined byscintillation counting. The amount of radioactivity in the cells addedto each well was determined independently, and was used to determine thefraction of applied cells that actually adhered to the monolayers ineach microliter well. In order to test the inhibitory activity ofsoluble lamp-1, purified soluble lamp-1 was dialyzed against PBS andaliquots, serially diluted in DME containing 5% fetal calf serum, wereadded to microtiter wells containing activated HUVEC monolayers. Afterincubation for 15 minutes at 4° C., the monolayers were used in adhesionassays as described above.

A substantial fraction of cells expressing recombinant cell surfacelamp-1 bound to HUVEC monolayers, which were induced to expressE-selectin by pre-treatment with IL1-β, whereas the same cells did notbind detectably to non-activated HUVECS (FIG. 2A). The parental SP cellsbound only modestly to activated HUVEC monolayers. Essentially all ofthe adhesion observed with the lamp-1 transfected cell lines isE-selectin-dependent, since binding of the cell line expressing thelargest amount of cell surface lamp-1 (transfected with pSRαlamp-1^(H),see FIG. 1) may be virtually completely blocked by pre-treatment of themonolayers with liposomes containing sialyl Lewis^(x) glycolipid (FIG.2B). Under the same conditions, control liposomes did not have aneffect.

Binding of SP cells to CHO cells expressing E-selectin was carried outin the same way except that the activation by IL-1β was omitted.Inhibition by sialyl Le^(x) glycolipid (Kameyama et al. Carbohydr. Res.209:C1-C4 (1991) incorporated herein by reference),NeuNAcα2→3Galβ1→4(Fucα1→3)GlcNAc.beta.1→3Galβ1→4Glc-Cer was testedexactly as described (Phillips et al., supra). Paragloboside,NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc-Cer was used as a controlglycolipid.

Similar results were obtained in experiments using CHO cells expressingE-selectin. Again, cells expressing increased levels of cell surfacelamp-1 bound to E-selectin-expressing CHO monolayers, but not to controlCHO monolayers that do not express E-selectin (FIG. 2A). The control,parent SP cells bound moderately to the CHO cell monolayer expressingE-selectin. When considered with the data shown in FIG. 1, theseobservations indicate that the ability to exhibit E-selectin-dependentadhesion in this static adhesion assay may be conferred upon the SPcells by affecting only a modest increase in cell surface lamp-1expression. The two-fold increase in cell surface lamp-1 expressionshown by pSVlamp-1-transfected cells, relative to the control SP cells(FIG. 1, compare SP vs pSVlamp-1) appears sufficient to enable the SPcells to efficiently adhere to E-selectin. Moreover, increased adhesionis seen when higher levels of cell surface lamp-1 are present. (forexample, compare binding and lamp-1 expression of pSVlamp-1transfectants; 48% bound, mean fluorescent intensity of ˜20, versusbinding and lamp-1 expression of pSRαlamp-1^(H) transfectants; ˜75%bound, mean fluorescent intensity of ˜60). These results establish thatthe lamp-1 on the cell surface carry ligands for E-selectin, and thedegree of binding is roughly proportional to the amount of lamp-1expressed on the cell surface.

EXAMPLE X Comparison of Cell Surface Sialyl Le^(x) Expression Among SPCells Expressing Different Amounts of Cell Surface Lamp-1

Cell surface lamp-1 molecules are heavily substituted with N- andO-linked oligosaccharide molecules that can terminate in the sialylLewis^(x) moiety (Lee et al., supra), an essential component of theoligosaccharide ligand for E-selectin. It therefore seemed possible thatincreased lamp-1 expression in turn would yield a concomitant increasein cell surface sialyl Lewis^(x) moieties (displayed by surfacelocalized lamp-1 molecules), and that this would conferE-selectin-dependent adhesion competence upon the lamp-1 transfectants.

A radioactive antibody binding assay was used to quantitate cell surfacesialyl Lewis^(x) termini on the cell lines that express recombinantlamp-1, and on the parental SP cells. The number of binding sites forthe monoclonal anti-sialyl Le^(x) antibody was measured as detailedpreviously (Saitoh et al., supra). At near-saturating levels ofanti-sialyl Lewis^(x) antibody, each of the cell lines that expressessupra-control level of lamp-1 also displayed a substantially higherlevel of cell surface sialyl Lewis^(x) immunoreactivity than the leveldisplayed by the control SP cells (FIG. 3). These data are mostconsistent with the hypothesis that the level of cell surface lamp-1expression can directly determine cell surface sialyl Lewis^(x)expression levels, and thus also E-selectin-dependent cell adhesion.

EXAMPLE XI Soluble Lamp-1 Can Inhibit E-Selectin Mediated Binding

The data obtained from the experiments detailed above support anessential role for lamp-1 in mediating E-selectin-dependent celladhesion of tumor cells, by functioning to present sialyl Lewis^(x) toE-selectin in a manner analogous to that proposed for L-selectin onleukocytes (Picker et al., Cell 66:921-933 (1991) incorporated herein byreference). It also suggests that soluble lamp-1 molecules that displaysialyl Lewis^(x) -terminated oligosaccharides may effectively disruptE-selectin-dependent cell adhesion by displacing cell-associated sialylLewis^(x) binding site(s) on E-selectin.

A soluble lamp-1 molecule that displays the sialyl Lewis^(x) moiety wasprepared and its ability to block E-selectin-dependent adhesion oflamp-1-expressing SP cells was tested. This reagent was prepared fromCHO cells stably transfected with a vector that directs the expressionof a soluble form of lamp-1, and with a vector that encodes a humanα(1,3)fucosyltransferase (Fuc-TIII) (Kukowska-Latallo et al., supra)capable of creating the sialyl Lewis^(x) determinant using endogenousCHO cell oligosaccharide precursors (Lowe et al., supra). Thetransfected cells were confirmed to express sialyl Le^(x) determinantsby immunofluorescence as described (Williams et al., supra). Thisrecombinant molecule was purified from media collected from these cellsusing wheat germ agglutinin and DEAE-Sephadex column chromatographyprocedures. The product of this purification consisted largely of asingle polypeptide (FIG. 4A) which by Western blotting reacted withanti-lamp-1 antibodies (FIG. 4B).

A soluble lamp-1 molecule lacking the sialyl Lewis^(x) determinant waspurified in an identical manner, using a control CHO cell line stablytransfected only with the vector that synthesizes soluble lamp-1molecules. This purified control protein also reacts with anti-lamp-1(FIG. 4B).

Using the adhesion assay previously shown to be E-selectin-dependent(FIG. 2B), it was determined that the concentration-dependent inhibitionof adhesion of pSRαlamp-1-transfected SP cells to activated HUVECmonolayers (FIG. 5). By contrast, the control, sialyl Lewis^(x)-negative lamp-1 molecule inhibited the binding minimally, even at aconcentration that for the sialyl Lewis^(x) -positive protein diminishedbinding to 20% of control levels. These results indicate that the sialylLewis^(x) determinant achieves a conformation on the soluble lamp-1glycoprotein that is recognized by E-selectin with an affinitysufficient to compete with the cell surface sialyl Lewis^(x)determinants that mediate adhesion to this selectin. These resultsfurther suggest that this reagent, and analogous ones, may prove usefulas therapeutic agents which block selectin-dependent inflammation ortumor metastasis.

Although the invention has been described with reference to thedisclosed embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2455 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 191..1438                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCGGGCGGGCTTCTTCGCTGCCGACGTACGACGAGTGGCCGGGCTCTTGCGTCTGG60                TAACGCGCTGTCTCTAACGCCAGCGCCGTCTCGCGCGCACTGCGCACAGACCACCCGCAG120               ACGCCCGGCAGTCCGCAGGCCCAAACGCGCACGCGACCCCGCTCTCCGCACCGTACCCGG180               CCGCCTCGGCATGGCGCCCCGCAGCGCCCGGCGACCCCTGCTGCTGCTA229                          MetAlaProArgSerAlaArgArgProLeuLeuLeuLeu                                       1510                                                                          CTGCCTGTTGCTGCTGCTCGGCCTCATGCATTGTCGTCAGCAGCCATG277                           LeuProValAlaAlaAlaArgProHisAlaLeuSerSerAlaAlaMet                              152025                                                                        TTTATGGTGAAAAATGGCAACGGGACCGCGTGCATAATGGCCAACTTC325                           PheMetValLysAsnGlyAsnGlyThrAlaCysIleMetAlaAsnPhe                              30354045                                                                      TCTGCTGCCTTCTCAGTGAACTACGACACCAAGAGTGGCCCCAAGAAC373                           SerAlaAlaPheSerValAsnTyrAspThrLysSerGlyProLysAsn                              505560                                                                        ATGACCTTTGACCTGCCATCAGATGCCACAGTGGTGCTCAACCGCAGC421                           MetThrPheAspLeuProSerAspAlaThrValValLeuAsnArgSer                              657075                                                                        TCCTGTGGAAAAGAGAACACTTCTGACCCCAGTCTCGTGATTGCTTTT469                           SerCysGlyLysGluAsnThrSerAspProSerLeuValIleAlaPhe                              808590                                                                        GGAAGAGGACATACACTCACTCTCAATTTCACGAGAAATGCAACACGT517                           GlyArgGlyHisThrLeuThrLeuAsnPheThrArgAsnAlaThrArg                              95100105                                                                      TACAGCGTTCAGCTCATGAGTTTTGTTTATAACTTGTCAGACACACAC565                           TyrSerValGlnLeuMetSerPheValTyrAsnLeuSerAspThrHis                              110115120125                                                                  CTTTTCCCCAATGCGAGCTCCAAAGAAATCAAGACTGTGGAATCTATA613                           LeuPheProAsnAlaSerSerLysGluIleLysThrValGluSerIle                              130135140                                                                     ACTGACATCAGGGCAGATATAGATAAAAAATACAGATGTGTTAGTGGC661                           ThrAspIleArgAlaAspIleAspLysLysTyrArgCysValSerGly                              145150155                                                                     ACCCAGGTCCACATGAACAACGTGACCGTAACGCTCCATGATGCCACC709                           ThrGlnValHisMetAsnAsnValThrValThrLeuHisAspAlaThr                              160165170                                                                     ATCCAGGCGTACCTTTCCAACAGCAGCTTCAGCAGGGGAGAGACACGC757                           IleGlnAlaTyrLeuSerAsnSerSerPheSerArgGlyGluThrArg                              175180185                                                                     TGTGAACAAGACAGGCCTTCCCCAACCACAGCGCCCCCTGCGCCACCC805                           CysGluGlnAspArgProSerProThrThrAlaProProAlaProPro                              190195200205                                                                  AGCCCCTCGCCCTCACCCGTGCCCAAGAGCCCCTCTGTGGACAAGTAC853                           SerProSerProSerProValProLysSerProSerValAspLysTyr                              210215220                                                                     AACGTGAGCGGCACCAACGGGACCTGCCTGCTGGCCAGCATGGGGCTG901                           AsnValSerGlyThrAsnGlyThrCysLeuLeuAlaSerMetGlyLeu                              225230235                                                                     CAGCTGAACCTCACCTATGAGAGGAAGGACAACACGACGGTGACAAGG949                           GlnLeuAsnLeuThrTyrGluArgLysAspAsnThrThrValThrArg                              240245250                                                                     CTTCTCAACATCAACCCCAACAAGACCTCGGCCAGCGGGAGCTGCGGC997                           LeuLeuAsnIleAsnProAsnLysThrSerAlaSerGlySerCysGly                              255260265                                                                     GCCCACCTGGTGACTCTGGAGCTGCACAGCGAGGGCACCACCGTCCTG1045                          AlaHisLeuValThrLeuGluLeuHisSerGluGlyThrThrValLeu                              270275280285                                                                  CTCTTCCAGTTCGGGATGAATGCAAGTTCTAGCCGGTTTTTCCTACAA1093                          LeuPheGlnPheGlyMetAsnAlaSerSerSerArgPhePheLeuGln                              290295300                                                                     GGAATCCAGTTGAATACAATTCTTCCTGACGCCAGAGACCCTGCCTTT1141                          GlyIleGlnLeuAsnThrIleLeuProAspAlaArgAspProAlaPhe                              305310315                                                                     AAAGCTGCCAACGGCTCCCTGCGAGCGCTGCAGGCCACAGTCGGCAAT1189                          LysAlaAlaAsnGlySerLeuArgAlaLeuGlnAlaThrValGlyAsn                              320325330                                                                     TCCTACAAGTGCAACGCGGAGGAGCACGTCCGTGTCACGAAGGCGTTT1237                          SerTyrLysCysAsnAlaGluGluHisValArgValThrLysAlaPhe                              335340345                                                                     TCAGTCAATATATTCAAAGTGTGGGTCCAGGCTTTCAAGGTGGAAGGT1285                          SerValAsnIlePheLysValTrpValGlnAlaPheLysValGluGly                              350355360365                                                                  GGCCAGTTTGGCTCTGTGGAGGAGTGTCTGCTGGACGAGAACAGCACG1333                          GlyGlnPheGlySerValGluGluCysLeuLeuAspGluAsnSerThr                              370375380                                                                     CTGATCCCCATCGCTGTGGGTGGTGCCCTGGCGGGGCTGGTCCTCATC1381                          LeuIleProIleAlaValGlyGlyAlaLeuAlaGlyLeuValLeuIle                              385390395                                                                     GTCCTCATCGCCTACCTCGTCGGCAGGAAGAGGAGTCACGCAGGCTAC1429                          ValLeuIleAlaTyrLeuValGlyArgLysArgSerHisAlaGlyTyr                              400405410                                                                     CAGACTATCTAGCCTGGTGCACGCAGGCACAGCAGCTGCAGGGGCCTCT1478                         GlnThrIle                                                                     415                                                                           GTTCCTTTCTCTGGGCTTAGGGTCCTGTCGAAGGGGAGGCACACTTTCTGCAAACGTTTC1538              TCAAATCTGCTTCATCCAATGTGAAGTTCATCTTGCAGCATTTACTATGCACAACAGAGT1598              AACTATCGAAATGACGGTGTTAATTTTGCTAACTGGGTTAAATATTTTGCTAACTGGTTA1658              AACATTAATATTTACCAAAGTAGGATTTTGAGGGTGGGGGTGCTCTCTCTGAGGGGGTGG1718              GGGTGCCGCTGTCTCTGAGGGGTGGGGGTGCCGCTGTCTGAGGGGTGGGGGTGCCGCTCT1778              CTCTGAGGGGGTGGGGGTGCCGCTTTCTCTGAGGGGGTGGGGGTGCCGCTCTCTCTGAGG1838              GGGTGGGGGTGCTGCTCTCTCCGAGGGGTGGAATGCCGCTGTCTCTGAGGGGTGGGGGTG1898              CCGCTCTAAATTGGCTCCATATCATTGAGTTTAGGGTTCTGGTGTTTGGTTTCTTCATTC1958              TTTACTGCACTCAGATTTAAGCCTTACAAAGGGAAACCTCTGGCCGTCACACGTAGGACG2018              CATGAAGGTCACTCGTGTGAGGCTGACATGCTCACACATTACAACAGTAGAGAGGGAAAA2078              TCCTAAGACAGAGGAACTCCAGAGATGAGTGTCTGGAGCGGCTTCAGTTCAGCTTTAAAG2138              GCCAGGACGCGCGACACGTGGCTGGCGGCCTCGTTCCAGTGGCGGCACGTCCTTGGCGTC2198              TCTAATGTCTGCAGCTCAAGGGCTGGCACTTTTTTAAATATAAAAATGGTGTTATTTTTA2258              TTTTTTTTTGTAAAGTGATTTTTGGTCTTCTGTTGACATTCGGGTGATCCTGTTCTGCGC2318              TGTGTACAATGTGAGATCGGTGCGTTCTCCTGATGTTTTGCCGTGGCTTGGGGATTGTAC2378              ACGGGACCAGCTCACGTAATGCATTGCCTGTAACAATGTAATAAAAAGCCTCTTTCTTTC2438              AAAAAAACCCCGAATTC2455                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 416 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaProArgSerAlaArgArgProLeuLeuLeuLeuLeuProVal                              151015                                                                        AlaAlaAlaArgProHisAlaLeuSerSerAlaAlaMetPheMetVal                              202530                                                                        LysAsnGlyAsnGlyThrAlaCysIleMetAlaAsnPheSerAlaAla                              354045                                                                        PheSerValAsnTyrAspThrLysSerGlyProLysAsnMetThrPhe                              505560                                                                        AspLeuProSerAspAlaThrValValLeuAsnArgSerSerCysGly                              65707580                                                                      LysGluAsnThrSerAspProSerLeuValIleAlaPheGlyArgGly                              859095                                                                        HisThrLeuThrLeuAsnPheThrArgAsnAlaThrArgTyrSerVal                              100105110                                                                     GlnLeuMetSerPheValTyrAsnLeuSerAspThrHisLeuPhePro                              115120125                                                                     AsnAlaSerSerLysGluIleLysThrValGluSerIleThrAspIle                              130135140                                                                     ArgAlaAspIleAspLysLysTyrArgCysValSerGlyThrGlnVal                              145150155160                                                                  HisMetAsnAsnValThrValThrLeuHisAspAlaThrIleGlnAla                              165170175                                                                     TyrLeuSerAsnSerSerPheSerArgGlyGluThrArgCysGluGln                              180185190                                                                     AspArgProSerProThrThrAlaProProAlaProProSerProSer                              195200205                                                                     ProSerProValProLysSerProSerValAspLysTyrAsnValSer                              210215220                                                                     GlyThrAsnGlyThrCysLeuLeuAlaSerMetGlyLeuGlnLeuAsn                              225230235240                                                                  LeuThrTyrGluArgLysAspAsnThrThrValThrArgLeuLeuAsn                              245250255                                                                     IleAsnProAsnLysThrSerAlaSerGlySerCysGlyAlaHisLeu                              260265270                                                                     ValThrLeuGluLeuHisSerGluGlyThrThrValLeuLeuPheGln                              275280285                                                                     PheGlyMetAsnAlaSerSerSerArgPhePheLeuGlnGlyIleGln                              290295300                                                                     LeuAsnThrIleLeuProAspAlaArgAspProAlaPheLysAlaAla                              305310315320                                                                  AsnGlySerLeuArgAlaLeuGlnAlaThrValGlyAsnSerTyrLys                              325330335                                                                     CysAsnAlaGluGluHisValArgValThrLysAlaPheSerValAsn                              340345350                                                                     IlePheLysValTrpValGlnAlaPheLysValGluGlyGlyGlnPhe                              355360365                                                                     GlySerValGluGluCysLeuLeuAspGluAsnSerThrLeuIlePro                              370375380                                                                     IleAlaValGlyGlyAlaLeuAlaGlyLeuValLeuIleValLeuIle                              385390395400                                                                  AlaTyrLeuValGlyArgLysArgSerHisAlaGlyTyrGlnThrIle                              405410415                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 210 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTTTTGCAAGGCTGTGGTCGGTGGTCATCAGTGCTCTTGACCCAGGTCCAGCGAGCCTTT60                TCCCTGGTGTTGCAGCTGTTGTTGTACCGCCGCCGTCGCCGCCGTCGCCGCCTGCTCTGC120               GGGGTCATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGC180               CTAGTCCTGGGTGAGTTGTCGGGCCCTCCC210                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 159 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATTTTTTTAAATGAATCCAGGAGCTGTGCGGTCTTATGCATTGGAACTTAATTTGACAGA60                TTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCAGATGAATTTCACAGTTCGCTATGA120               AACTACAAATAAAACTTATGTAAGTATATATTTGGGTTT159                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 254 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAAATTTCTATTTCTTTTAGAAAACTGTAACCATTTCAGACCATGGCACTGTGACATATA60                ATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGACCTG120               GCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAAATGACAGCGTCT180               CATTTTCCTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGTAACC240               TTAAGAATGGATTT254                                                             (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 199 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TTGTTAATCTTGTTTTATAGGAATTCTTACTGTTGATGAACTTTTGGCCATCAGAATTCC60                ATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATGTTGTCCA120               ACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGG180               TGAGTAACAACAGATTTTT199                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 225 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCCCTTTTCGCTTGTTTTAGAGTTCCTGTGTGATAAAGACAAAACTTCAACAGTGGCACC60                CACCATACACACCACTGTGCCATCTCCTACTACAACACCTACTCCAAAGGAAAAACCAGA120               ACCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGCGTGCTACCATGGGGCT180               GCAGCTGAACATCACTCAGGATAAGGTATAGGTGTCTATCTTTAT225                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 163 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCTTTCTTCTTCTCCTGAAGGTTGCTTCAGTTATTAACATCAACCCCAATACAACTCACT60                CCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAGT120               ATCTAGACTTTGTCTTTGCTGTGGTGAGTAACAACAGATTTTT163                                (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGAAGCTCTTTTTCAAACAGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCA60                GCATGTATTTGGTTAATGGCTCCGGTAAGCAAAGCACTGGACCT104                               (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 205 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCTGTTTCTTTTCTTTGAAGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGC60                CCCCCTGGGAAGTTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATT120               TCAGATAAATACCTTTGATCTAAGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTC180               TACAGGTAAGAATCAAGCAAACTTC205                                                  (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 687 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TGTCCTTTCTCCACATCTAGCTCAAGACTGCAGTGCAGATGACGACAACTTCCTTGTGCC60                CATAGCGGTGGGAGCTGCCTTGGCAGGAGTACTTATTCTAGTGTTGCTGGCTTATTTTAT120               TGGTCTCAAGCACCATCATGCTGGATATGAGCAATTTTAGAATCTGCAACCTGATTGATT180               ATATAAAAATACATGCAAATAACAAGATTTTCTTACCTCTCAGTTGTTGAAACACTTTGC240               TTCTTAAAATTGATATGTTGAAACTTTAATTCTTTTATCAATCCCAGCATTTTGAGATCA300               GTCTTTATTAATAAAACCTGTTCTCTTTAATCAGCTTAAAATCCAAAGTGTCATATTTAC360               TGGTCCTGGAGACAAACTTGTTCAAAAGAACATCAACGTGCAATGTTTTAAGGGTCTATC420               TTAAGGAAGCCCTGGCCAAATTTTGACCTAACTTGAAGTATCCTTGAACTTATTAACATG480               GCCATTATAAGAATAAAATATGTAGTTGTGTCTTAATGGAATTAATAAATGTCATTTCAC540               TACTGGTGTTCTGTTTCAATCTATAAGGACTATAGTGATTTAAACTCATCAATGTGCCTT600               TGCATAAAGTTCATTAAATAAATATTGATGTGGTATAAATGCCCATCAGATATGCTTAAA660               CTTGGTTTTCAGTTGAATGAAGTAGAG687                                                (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TCAAGTACTCTTGAAGTCATGATTGCTTCA30                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TGAAGTACTAACTTAAAGGATGTAAGAAGGCTT33                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TTTGAATTCCTCGCGCCATGGCGCC25                                                   (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AAAGGTACCTAGCTGTTCTCGTCCAGCAG29                                               (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 410 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetValCysPheArgLeuPheProValProGlySerGlyLeuValLeu                              151015                                                                        ValLeuValCysLeuGlyAlaValArgSerTyrAlaLeuGluLeuAsn                              202530                                                                        LeuThrAspSerGluAsnAlaThrCysLeuTyrAlaLysTrpGlnMet                              354045                                                                        AsnPheThrValArgTyrGluThrThrAsnLysThrTyrLysThrVal                              505560                                                                        ThrIleSerAspHisGlyThrValThrTyrAsnGlySerIleCysGly                              65707580                                                                      AspAspGlnAspGlyProLysIleAlaValGlnPheGlyProGlyPhe                              859095                                                                        SerTrpIleAlaAsnPheThrLysAlaAlaSerThrTyrSerAsnAsp                              100105110                                                                     SerValSerPheSerTyrAsnThrGlyAspAsnThrThrPheProAsp                              115120125                                                                     AlaGluAspLysGlyIleLeuThrValAspGluLeuLeuAlaIleArg                              130135140                                                                     IleProLeuAsnAspLeuPheArgCysAsnSerLeuSerThrLeuGlu                              145150155160                                                                  LysAsnAspValValGlnHisTyrTrpAspValLeuValGlnAlaPhe                              165170175                                                                     ValGlnAsnGlyThrValSerThrAsnGluPheLeuCysAspLysAsp                              180185190                                                                     LysThrSerThrValAlaProThrIleHisThrThrValProSerPro                              195200205                                                                     ThrThrThrProThrProLysGluLysProGluProGlyThrTyrSer                              210215220                                                                     ValAsnAsnGlyAsnAspThrCysLeuLeuAlaThrMetGlyLeuGln                              225230235240                                                                  LeuAsnIleThrGlnAspLysValAlaSerValIleAsnIleAsnPro                              245250255                                                                     AsnThrThrHisSerThrGlySerCysArgSerHisThrAlaLeuLeu                              260265270                                                                     ArgLeuAsnSerSerThrIleLysTyrLeuAspPheValPheAlaVal                              275280285                                                                     LysAsnGluAsnArgPheTyrLeuLysGluValAsnIleSerMetTyr                              290295300                                                                     LeuValAsnGlySerValPheSerIleAlaAsnAsnAsnLeuSerTyr                              305310315320                                                                  TrpAspAlaProLeuGlySerSerTyrMetCysAsnLysGluGlnThr                              325330335                                                                     ValSerValSerGlyAlaPheGlnIleAsnThrPheAspLeuArgVal                              340345350                                                                     GlnProPheAsnValThrGlnGlyLysTyrSerThrAlaGlnAspCys                              355360365                                                                     SerAlaAspAspAspAsnPheLeuValProIleAlaValGlyAlaAla                              370375380                                                                     LeuAlaGlyValLeuIleLeuValLeuLeuAlaTyrPheIleGlyLeu                              385390395400                                                                  LysHisHisHisAlaGlyTyrGluGlnPhe                                                405410                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 353 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AlaMetPheMetValLysAsnGlyAsnGlyThrAlaCysIleMetAla                              151015                                                                        AsnPheSerAlaAlaPheSerValAsnTyrAspThrLysSerGlyPro                              202530                                                                        LysAsnMetThrPheAspLeuProSerAspAlaThrValValLeuAsn                              354045                                                                        ArgSerSerCysGlyLysGluAsnThrSerAspProSerLeuValIle                              505560                                                                        AlaPheGlyArgGlyHisThrLeuThrLeuAsnPheThrArgAsnAla                              65707580                                                                      ThrArgTyrSerValGlnLeuMetSerPheValTyrAsnLeuSerAsp                              859095                                                                        ThrHisLeuPheProAsnAlaSerSerLysGluIleLysThrValGlu                              100105110                                                                     SerIleThrAspIleArgAlaAspIleAspLysLysTyrArgCysVal                              115120125                                                                     SerGlyThrGlnValHisMetAsnAsnValThrValThrLeuHisAsp                              130135140                                                                     AlaThrIleGlnAlaTyrLeuSerAsnSerSerPheSerArgGlyGlu                              145150155160                                                                  ThrArgCysGluGlnAspArgProSerProThrThrAlaProProAla                              165170175                                                                     ProProSerProSerProSerProValProLysSerProSerValAsp                              180185190                                                                     LysTyrAsnValSerGlyThrAsnGlyThrCysLeuLeuAlaSerMet                              195200205                                                                     GlyLeuGlnLeuAsnLeuThrTyrGluArgLysAspAsnThrThrVal                              210215220                                                                     ThrArgLeuLeuAsnIleAsnProAsnLysThrSerAlaSerGlySer                              225230235240                                                                  CysGlyAlaHisLeuValThrLeuGluLeuHisSerGluGlyThrThr                              245250255                                                                     ValLeuLeuPheGlnPheGlyMetAsnAlaSerSerSerArgPhePhe                              260265270                                                                     LeuGlnGlyIleGlnLeuAsnThrIleLeuProAspAlaArgAspPro                              275280285                                                                     AlaPheLysAlaAlaAsnGlySerLeuArgAlaLeuGlnAlaThrVal                              290295300                                                                     GlyAsnSerTyrLysCysAsnAlaGluGluHisValArgValThrLys                              305310315320                                                                  AlaPheSerValAsnIlePheLysValTrpValGlnAlaPheLysVal                              325330335                                                                     GluGlyGlyGlnPheGlySerValGluGluCysLeuLeuAspGluAsn                              340345350                                                                     Ser                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 380 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      MetAlaProArgSerAlaArgArgProLeuLeuLeuLeuLeuProVal                              151015                                                                        AlaAlaAlaArgProHisAlaLeuSerSerAlaAlaMetPheMetVal                              202530                                                                        LysAsnGlyAsnGlyThrAlaCysIleMetAlaAsnPheSerAlaAla                              354045                                                                        PheSerValAsnTyrAspThrLysSerGlyProLysAsnMetThrPhe                              505560                                                                        AspLeuProSerAspAlaThrValValLeuAsnArgSerSerCysGly                              65707580                                                                      LysGluAsnThrSerAspProSerLeuValIleAlaPheGlyArgGly                              859095                                                                        HisThrLeuThrLeuAsnPheThrArgAsnAlaThrArgTyrSerVal                              100105110                                                                     GlnLeuMetSerPheValTyrAsnLeuSerAspThrHisLeuPhePro                              115120125                                                                     AsnAlaSerSerLysGluIleLysThrValGluSerIleThrAspIle                              130135140                                                                     ArgAlaAspIleAspLysLysTyrArgCysValSerGlyThrGlnVal                              145150155160                                                                  HisMetAsnAsnValThrValThrLeuHisAspAlaThrIleGlnAla                              165170175                                                                     TyrLeuSerAsnSerSerPheSerArgGlyGluThrArgCysGluGln                              180185190                                                                     AspArgProSerProThrThrAlaProProAlaProProSerProSer                              195200205                                                                     ProSerProValProLysSerProSerValAspLysTyrAsnValSer                              210215220                                                                     GlyThrAsnGlyThrCysLeuLeuAlaSerMetGlyLeuGlnLeuAsn                              225230235240                                                                  LeuThrTyrGluArgLysAspAsnThrThrValThrArgLeuLeuAsn                              245250255                                                                     IleAsnProAsnLysThrSerAlaSerGlySerCysGlyAlaHisLeu                              260265270                                                                     ValThrLeuGluLeuHisSerGluGlyThrThrValLeuLeuPheGln                              275280285                                                                     PheGlyMetAsnAlaSerSerSerArgPhePheLeuGlnGlyIleGln                              290295300                                                                     LeuAsnThrIleLeuProAspAlaArgAspProAlaPheLysAlaAla                              305310315320                                                                  AsnGlySerLeuArgAlaLeuGlnAlaThrValGlyAsnSerTyrLys                              325330335                                                                     CysAsnAlaGluGluHisValArgValThrLysAlaPheSerValAsn                              340345350                                                                     IlePheLysValTrpValGlnAlaPheLysValGluGlyGlyGlnPhe                              355360365                                                                     GlySerValGluGluCysLeuLeuAspGluAsnSer                                          370375380                                                                     __________________________________________________________________________

What is claimed:
 1. A lamp-1 polypeptide having the amino acid sequenceshown in SEQ ID NO.: 17, wherein the lamp-1 polypeptide is glycosylatedwith sialyl Le^(x), soluble in an aqueous solvent and binds toE-selectin.
 2. The lamp-1 polypeptide of claim 1 produced by a methodcomprising the steps of:a. inserting into a suitable vector a nucleicacid molecule encoding the lamp-1 polypeptide of claim 1; b.transfecting the resulting vector into a suitable host cell thatexpresses fucosyltransferase enzyme; c. culturing the resulting hostcell under conditions suitable for the expression of the lamp-1polypeptide; and d. recovering the lamp-1 polypeptide so produced.
 3. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the lamp-1 polypeptide of claim
 1. 4. A lamp-1 polypeptidehaving the amino acid sequence shown in SEQ ID NO.: 18, wherein thelamp-1 polypeptide is glycosylated with sialyl Le^(x), soluble in anaqueous solvent and binds to E-selectin.
 5. The lamp-1 polypeptide ofclaim 4 produced by the method comprising:a. inserting into a suitablevector a nucleic acid molecule encoding the lamp-1 polypeptide of claim4; b. transfecting the resulting vector into a suitable host cell thatexpresses fucosyltransferase enzyme; c. culturing the resulting hostcell under conditions suitable for the expression of the lamp-1polypeptide; and d. recovering the lamp-1 polypeptide so produced.
 6. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the lamp-1 polypeptide of claim 4.